Fig 3.

Knockdown performance characteristics of siRNAs targeting IκBs. (A) Diagram of the three IκB mRNAs showing regions targeted by the siRNAs (black) and RT-PCR probes used (arrows). (B) Cell viability following transfection with IκB siRNAs, determined by MTS assay. Little to no decrease in cell viability was observed over 72 h. (C) IκB mRNA levels after siRNA transfection. Individual IκB mRNAs were quantitated by qRT-PCR at serial times following transfection with IκB siRNAs and normalized to endogenous GAPDH. All mRNAs were knocked down substantially, with a nadir for each IκB mRNA at 12 h. (D) The half-life for each protein was calculated determining the slope of each protein knockdown. IκBα had a short half-life (5 h), compared to IκBβ (17 h) and IκBε (33 h). (E) IκB protein levels after siRNA transfection. Individual IκB proteins were quantitated by immunoblotting using specific monoclonal antibodies and densitometry at serial times following transfection with IκB siRNAs and normalized to β-actin. Each subpanel lists the specific siRNA at the top of the subpanel. In contrast to the results for the IκB mRNA levels, the IκB protein levels reached nadirs at different times following transfection, exhibiting substantially different half-lives and different recovery kinetics. IκBα protein reached a nadir at 12 h, similar to that seen for IκBα mRNA, and started to recover back to baseline relatively quickly. In contrast, IκBβ and IκBε proteins did not reach a nadir until 48 h after transfection and then recovered slowly. These results are consistent with those seen following treatment with TNF-α (Fig. 1). The individual siRNAs exhibited little off-target effect for the other nontargeted IκBs. The half-life for each protein was calculated determining the slope of each protein knockdown (D).